Molecular dissection of the genes involved in thermotolerance and spore pigment systhesis in Aspergillus fumigatus and their significance in virulence. Aspergillus is one of the most common fungal pathogens affecting neutropenic patients and other types of immunocompromised individuals such as those with Chronic Granulomatous Disease of Childhood. Among a dozen species of Aspergillus reported to cause infection in humans, A. fumigatus is the most common species reported to cause invasive aspergillosis. All Aspergillus species propagate by conidia (spores), which humans encounter daily through inhalation. We have focused our attention on the molecular genetic aspects of conidial pigment biosynthesis since pigment is one of the visible components of the wall that protect conidia. In the previous years we have characterized six developmentally associated genes involved in pentaketide melanin synthesis which are clustered within a 19kb fragment of A. fumigatus genomic DNA. Furthermore, we have shown that the conidial pigment synthetic pathway plays an important role in pathogenesis. In 2001-2003, we identified the product of one of the six clustered genes, AYG1, a novel protein which shortens the length of the polyketide carbon skeleton from a heptaketide to pentaketide. We have also purified the Ayg1 protein and identified its reaction mechanism and properties. The purified Ayg1p converted the heptaketide YWA1 to the pentaketide T4HN with release of the deketide acetoacetate, which was confirmed by LC-MS analysis as its o-(2,3,4,5,6-pentafluorobenzyl)oxime. This conversion was strongly inhibited by serine protease inhibitor 3,4-dichloroisocoumarin which modifies the serine residue in the catalytic center. Thus, it was concluded that Ayg1p catalyzes the hydrolytic cleavage of C-C bond between naphthalene ring and the side chain 1'-carbonyl of YWA1 which is attacked by hydroxy anion of the serine residue of the active center. Then, o-acetoacetylated Ayg1p is hydrolyzed to release acetoacetate. This year, we have identified the catalytic triad Asp-His-Ser by site directed mutagenesis. Sigle substitution mutations, S257A, D352A, and H380A resulted in a complete loss of Ayg1p activity. We also completed idetification of the thermotolerance gene THTA of A. fumigatus and developed an efficient transformation system via Agrobacterium tumefecience (ATMT). ATMT has proven to be an efficient molecular tool for insertional mutagenesis as well as gene disruption by homologous recombination.